Multiple Sequence Alignment Analysis Research Articles

Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants

BackgroundHigh-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants.MethodsThe complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants.ResultsAltogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation.ConclusionsNucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Genome-wide characterization of SINA E3 ubiquitin ligase family members and their expression profiles in response to various abiotic stresses and hormones in kiwifruit

SINA (Seven in absentia) proteins in the subtype of E3 ubiquitin ligase family have important functions in regulating the growth and development as well as in response to abiotic and biotic stresses in plants. However, the characteristics and possible functions of SINA family proteins in kiwifruit are not studied. In this research, a total number of 11 AcSINA genes in the kiwifruit genome were identified. Chromosome location and multiple sequence alignment analyses indicated that they were unevenly distributed on 10 chromosomes and all contained the typical N-terminal RING domain and C-terminal SINA domain. Phylogenetic, gene structure and collinear relationship analyses revealed that they were highly conserved with the same gene structure, and have gone through segmental duplication events. Expression pattern analyses demonstrated that all AcSINAs were ubiquitously expressed in roots, stems and leaves, and were responsive to different abiotic and plant hormone treatments with overlapped but distinct expression patterns. Further yeast two-hybrid and Arabidopsis transformation analyses demonstrated most AcSINAs interacted with itself or other AcSINA members to form homo- or heterodimers, and ectopic expression of AcSINA2 in Arabidopsis led to hypersensitive growth phenotype of transgenic seedlings to ABA treatment. Our results reveal that AcSINAs take part in the response to various abiotic stresses and hormones, and provide important information for the functional elucidation of AcSINAs in vine fruit plants.

Mutations of SARS-CoV-2 Structural Proteins in the Alpha, Beta, Gamma, and Delta Variants: Bioinformatics Analysis.

COVID-19 and Middle East Respiratory Syndrome are two pandemic respiratory diseases caused by coronavirus species. The novel disease COVID-19 caused by SARS-CoV-2 was first reported in Wuhan, Hubei Province, China, in December 2019, and became a pandemic within 2-3 months, affecting social and economic platforms worldwide. Despite the rapid development of vaccines, there have been obstacles to their distribution, including a lack of fundamental resources, poor immunization, and manual vaccine replication. Several variants of the original Wuhan strain have emerged in the last 3 years, which can pose a further challenge for control and vaccine development. The aim of this study was to comprehensively analyze mutations in SARS-CoV-2 variants of concern (VoCs) using a bioinformatics approach toward identifying novel mutations that may be helpful in developing new vaccines by targeting these sites. Reference sequences of the SARS-CoV-2 spike (YP_009724390) and nucleocapsid (YP_009724397) proteins were compared to retrieved sequences of isolates of four VoCs from 14 countries for mutational and evolutionary analyses. Multiple sequence alignment was performed and phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA (version 6). Mutations in amino acid sequences were analyzed using the MultAlin online tool (version 5.4.1). Among the four VoCs, a total of 143 nonsynonymous mutations and 8 deletions were identified in the spike and nucleocapsid proteins. Multiple sequence alignment and amino acid substitution analysis revealed new mutations, including G72W, M2101I, L139F, 209-211 deletion, G212S, P199L, P67S, I292T, and substitutions with unknown amino acid replacement, reported in Egypt (MW533289), the United Kingdom (MT906649), and other regions. The variants B.1.1.7 (Alpha variant) and B.1.617.2 (Delta variant), characterized by higher transmissibility and lethality, harbored the amino acid substitutions D614G, R203K, and G204R with higher prevalence rates in most sequences. Phylogenetic analysis among the novel SARS-CoV-2 variant proteins and some previously reported β-coronavirus proteins indicated that either the evolutionary clade was weakly supported or not supported at all by the β-coronavirus species. This study could contribute toward gaining a better understanding of the basic nature of SARS-CoV-2 and its four major variants. The numerous novel mutations detected could also provide a better understanding of VoCs and help in identifying suitable mutations for vaccine targets. Moreover, these data offer evidence for new types of mutations in VoCs, which will provide insight into the epidemiology of SARS-CoV-2.

Major histocompatibility complex class IIα and IIβ of pufferfish (Takifugu obscurus): Identification and functional characterization in response to bacterial challenge.

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf-MHC IIα) and MHC IIβ (Pf-MHC IIβ) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf-MHC IIα and Pf-MHC IIβ were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf-MHC IIα or Pf-MHC IIβ contained a signal peptide, an α1/β1 domain, an α2/β2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf-MHC IIα and Pf-MHC IIβ molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf-MHC IIα and Pf-MHC IIβ was in the lymphocyte membrane and cytoplasm. qRT-PCR results showed that Pf-MHC IIα and Pf-MHC IIβ expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf-MHC IIα and Pf-MHC IIβ were significantly up-regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf-MHC IIα or Pf-MHC IIβ neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF-α, IL-1β and IL-10). Overall, it is speculated that Pf-MHC IIα and Pf-MHC IIβ may play an important role in the host response against A. hydrophila in pufferfish.

Molecular characterization and 3D Modeling of p62 in Grass Carp (Ctenopharyngodon Idella)

P62 is a ubiquitin-binding protein that plays a key role in biological pathways including autophagy and protein degradation. In this study, we analyzed the protein sequence of grass carp p62 (gcp62) and its 3D structure was predicated. The full-length cDNA of gcp62 was 1425 bp, and encoded a 474-aa protein. Multiple sequence alignment analysis showed that gcp62 shared 40.82~41.80% amino acid identities with its orthologues of human, mouse and tropical clawed frog, as well as relatively high identities (63.28 and 78.71% respectively) with yellow catfish and zebrafish p62. The phylogenetic analysis showed that gcp62 has close relationship with its orthologues in common carp and zebrafish, but differs from its homologs in mammalian species. Like in mammals, gcp62 was predicted to contain three conserved domains, the N-terminal Phox and Bem1p (PB1) domain, the ZZ domain and the C-terminal ubiquitin-associated (UBA)domain. This is the first time to construct the 3D structure of fish p62 and the high similarity of p62 structure between human and fish indicates the functional conservation in vertebrates.

Genome-wide identification and expression analysis of VQ gene family under abiotic stress in Coix lacryma-jobi L.

BackgroundValine-glutamine (VQ) proteins are non-specific plant proteins that have a highly conserved motif: FxxhVQxhTG. These proteins are involved in the development of various plant organs such as seeds, hypocotyls, flowers, leaves and also play a role in response to salt, drought and cold stresses. Despite their importance, there is limited information available on the evolutionary and structural characteristics of VQ family genes in Coix lacryma-jobi.ResultsIn this study, a total of 31 VQ genes were identified from the coix genome and classified into seven subgroups (I–VII) based on phylogenetic analysis. These genes were found to be unevenly distributed on 10 chromosomes. Gene structure analysis revealed that these genes had a similar type of structure within each subfamily. Moreover, 27 of ClVQ genes were found to have no introns. Conserved domain and multiple sequence alignment analysis revealed the presence of a highly conserved sequences in the ClVQ protein. This research utilized quantitative real-time PCR (qRT-PCR) and promoter analysis to investigate the expression of ClVQ genes under different stress conditions. Results showed that most ClVQ genes responded to polyethylene glycol, heat treatment, salt, abscisic acid and methyl jasmonate treatment with varying degrees of expression. Furthermore, some ClVQ genes exhibited significant correlation in expression changes under abiotic stress, indicating that these genes may act synergistically in response to adversarial stress. Additionally, yeast dihybrid verification revealed an interaction between ClVQ4, ClVQ12, and ClVQ26.ConclusionsThis study conducted a genome-wide analysis of the VQ gene family in coix, including an examination of phylogenetic relationships, conserved domains, cis-elements and expression patterns. The goal of the study was to identify potential drought resistance candidate genes, providing a theoretical foundation for molecular resistance breeding.

Genome-Wide Analysis of Strictosidine Synthase-like Gene Family Revealed Their Response to Biotic/Abiotic Stress in Poplar.

The strictosidine synthase-like (SSL) gene family is a small plant immune-regulated gene family that plays a critical role in plant resistance to biotic/abiotic stresses. To date, very little has been reported on the SSL gene in plants. In this study, a total of thirteen SSLs genes were identified from poplar, and these were classified into four subgroups based on multiple sequence alignment and phylogenetic tree analysis, and members of the same subgroup were found to have similar gene structures and motifs. The results of the collinearity analysis showed that poplar SSLs had more collinear genes in the woody plants Salix purpurea and Eucalyptus grandis. The promoter analysis revealed that the promoter region of PtrSSLs contains a large number of biotic/abiotic stress response elements. Subsequently, we examined the expression patterns of PtrSSLs following drought, salt, and leaf blight stress, using RT-qPCR to validate the response of PtrSSLs to biotic/abiotic stresses. In addition, the prediction of transcription factor (TF) regulatory networks identified several TFs, such as ATMYB46, ATMYB15, AGL20, STOP1, ATWRKY65, and so on, that may be induced in the expression of PtrSSLs in response to adversity stress. In conclusion, this study provides a solid basis for a functional analysis of the SSL gene family in response to biotic/abiotic stresses in poplar.

Discovery of andrographolide hit analog as a potent cyclooxygenase-2 inhibitor through consensus MD-simulation, electrostatic potential energy simulation and ligand efficiency metrics

Cyclooxygenase-2 (COX-2) is the key enzyme responsible for the conversion of arachidonic acid to prostaglandins that display pro-inflammatory properties and thus, it is a potential target protein to develop anti-inflammatory drugs. In this study, chemical and bio-informatics approaches have been employed to find a novel potent andrographolide (AGP) analog as a COX-2 inhibitor having better pharmacological properties than aspirin and rofecoxib (controls). The full amino acid sequenced human Alpha fold (AF) COX-2 protein (604AA) was selected and validated for its accuracy against the reported COX-2 protein structures (PDB ID: 5F19, 5KIR, 5F1A, 5IKQ and 1V0X) followed by multiple sequence alignment analysis to establish the sequence conservation. The systematic virtual screening of 237 AGP analogs against AF-COX-2 protein yielded 22 lead compounds based on the binding energy score (< − 8.0 kcal/mol). These were further screened out to 7 analogs by molecular docking analysis and investigated further for ADMET prediction, ligand efficiency metrics calculations, quantum mechanical analysis, MD simulation, electrostatic potential energy (EPE) docking simulation, and MM/GBSA. In-depth analysis revealed that AGP analog A3 (3-[2-[(1R,4aR,5R,6R,8aR)-6-hydroxy-5,6,8a-trimethyl-2-methylidene-3,4,4a,5,7,8-hexahydro-1H-naphthalen-1-yl]ethylidene]-4-hydroxyoxolan-2-one) forms the most stable complex with the AF-COX-2 showing the least RMSD value (0.37 ± 0.03 nm), a good number of hydrogen bonds (protein–ligand H-bond = 11, and protein H-bond = 525), minimum EPE score (− 53.81 kcal/mol), and lowest MM-GBSA before and after simulation (− 55.37 and − 56.25 kcal/mol, respectively) value compared to other analogs and controls. Thus, we suggest that the identified A3 AGP analog could be developed as a promising plant-based anti-inflammatory drug by inhibiting COX-2.

Characterization, Evolutionary Analysis, and Expression Pattern Analysis of the Heat Shock Transcription Factors and Drought Stress Response in Heimia myrtifolia

Heat shock transcription factors (HSFs) are among the most important regulators of plant responses to abiotic stimuli. They play a key role in numerous transcriptional regulatory processes. However, the specific characteristics of HSF gene family members and their expression patterns in different tissues and under drought stress have not been precisely investigated in Heimia myrtifolia. This study analyzed transcriptome data from H. myrtifolia and identified 15 members of the HSF family. Using a phylogenetic tree, these members were classified into three major classes and fifteen groups. The amino acid physicochemical properties of these members were also investigated. The results showed that all HmHSF genes are located in the nucleus, and multiple sequence alignment analysis revealed that all HmHSF proteins have the most conserved DBD structural domains. Interestingly, a special HmHSF15 protein was found in the three-dimensional structure of the protein, which has a conserved structural domain that performs a function in addition to the unique structural domain of HSF proteins, resulting in a three-dimensional structure for HmHSF15 that is different from other HmHSF proteins. GO enrichment analysis shows that most HmHSFA-like genes are part of various biological processes associated with abiotic stresses. Finally, this study analyzed the tissue specificity of HmHSF genes in different parts of H. myrtifolia by qRT-PCR and found that HmHSF genes were more abundantly expressed in roots than in other tissues, and HmHSF05, HmHSF12, and HmHSF14 genes were different from other HSF genes, which could be further analyzed to verify their functionality. The results provide a basis for analyzing the functions of HmHSF genes in H. myrtifolia and help to explore the molecular regulatory mechanism of HmHSF in response to drought stress.

Expression profiling and function analysis highlight the positive involvement of sweet cherry PavTCP17 in regulating flower bud dormancy

The TCP transcription factors function diversely in plant growth, development and stress response. The flower bud dormancy was particularly important for the yield formation of fruit trees. However, there has been no in-depth study on the involvement of TCP genes in regulating flower bud dormancy in sweet cherry so far. In the present study, a total of 18 TCP genes were obtained based on the whole genome, which were divided into two subfamilies and three subclasses (PCF, CIN, CYC/TB1) by multiple sequence alignment and evolutionary analysis. Four TCP genes targeted by miR319 were identified in the degradome results. Tissue-specific expression of TCP gene was verified based on transcriptome data and qRT-PCR. The expression patterns of PavTCPs under cold treatment in dormant buds of two cold-requiring sweet cherry cultivars were analyzed, among which PavTCP17 was significantly positively correlated with the gene expressions of gibberellin (GA) degradation and abscisic acid (ABA) synthesis. The abundance of PavTCP17 gene expression was significantly reduced after low cold-requiring cultivars met its chilling demand. The qRT-PCR quantification also showed a decreasing trend of PavTCP17 expression level during blooming process. To further unravel the role of PavTCP17 gene in positively regulating floral bud dormancy, transgenic Arabidopsis overexpressed PavTCP17 was obtained by genetic transformation. Compared with the WT, the number of main branches and rosette leaves was significantly reduced in PavTCP17-overexpressing lines. Moreover, the outgrowth of lateral buds of the PavTCP17-overexpressing lines were remarkably inhibited, mirroring the formation of bud dormancy. Furthermore, PavTCP17 might directly bind to PavNCED3 promoter of sweet cherry, suggesting the implication of PavTCP17 in promoting ABA synthesis, which was essential for bud dormancy. Our study bettered the understanding of TCP gene functions and revealed the mechanism by which PavTCP17 positively regulates flower bud dormancy in sweet cherry.

Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus.

Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones. In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16°C and partially purified. These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

Abstract 3761: Mediators of RNA sorting and export in non-small cell lung cancer derived extracellular vesicles

Abstract Extracellular vesicle (EV) mediated transfer of biologically functional molecules between cancer cells and the surrounding environment significantly impacts cancer progression. Our previous study established that non-small cell lung cancer (NSCLC) cell-derived EV-RNA is an important mediator of this communication, since it significantly augments the invasive potential of recipient non-tumorigenic cells (BEAS2Bs). This led us to determine the EV-RNA cargo that is specific to NSCLC EVs. Our RNA sequencing analysis revealed preferential enrichment of specific repertoires of RNA in NSCLC EVs amongst which miR-10b, miR-100 and miR-155 show synergistic effects with regard to an invasive phenotype elicited in recipient BEAS2B cells. Interestingly, our RNA sequencing analysis also revealed a global decrease in specific subsets of microRNAs (miRNAs) in NSCLC (H358 and Calu6) cells in comparison to non-tumorigenic BEAS2B cells. In contrast, the dysregulated miRNAs were found to be exclusively abundant in the respective NSCLC cell derived EVs. This suggests a precise and active mechanism of RNA sorting and loading into EVs. A cohort of dysregulated miRNAs was shortlisted, and their cellular and EV expression was validated using RT-qPCR and flow cytometry analysis by conjugating each one of the candidates to a fluorophore. After confirming that inclusion of a fluorophore to the RNA does not disrupt RNA sorting into the EVs, we transfected the cohort of shortlisted miRNAs including miR-122, miR-451a, miR-486, miR-150, miR-200b, and miR-142 each conjugated with a separate fluorophore and evaluated the dynamics of export. As a control, one cell-retained miRNA was also transfected with the pool of candidate miRNAs and the fluorescence of transfected cells was monitored at different times. Our results indicate that of all miRNAs, the fluorescence specific to miR-122 and miR-150 diminishes in less than 72 hours while others are retained longer, indicating that all miRNAs are not sorted or exported similarly. EV-RNA release was validated by treating the cells with an inhibitor of EV biogenesis i.e., nSMase inhibitor Gw4869. The inhibitor caused the cells to retain the candidate fluorescence corresponding to the respective EV-RNAs. To investigate sequence similarities between the miRNAs exclusively enriched in EVs of NSCLC cells, multiple sequence alignment analysis (STREME) was conducted, and specific motif sequences overrepresented in EVs and lost in the cells were shortlisted. Multiple lines of evidence indicate that dysregulated RNA subsets are not only causal agents of cancer progression but also contribute to poor patient outcome. Hence, there is a critical need to evaluate EV export of these RNAs as a potential mechanism of RNA dysregulation in cancer. Resulting data from this study is expected to not only reveal mechanistic details of EV-RNA export but will also inform future therapeutic targets. Citation Format: Humna Hasan, Nadia A. Lanman, Sagar Utturkar, Jeremiah J. Jauch, Ikjot S. Sohal, Zulaida Soto-Vargas, Andrea L. Kasinski. Mediators of RNA sorting and export in non-small cell lung cancer derived extracellular vesicles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3761.

Retrospective investigation of the origin and epidemiology of the dengue outbreak in Yunnan, China from 2017 to 2018.

Since 2013, a dengue epidemic has broken out in Yunnan, China and neighboring countries. However, after the COVID-19 pandemic in 2019, the number of dengue cases decreased significantly. In this retrospective study, epidemiological and genetic diversity characterizations of dengue viruses (DENV) isolated in Yunnan between 2017 and 2018 were performed. The results showed that the dengue outbreak in Yunnan from 2017 to 2018 was mainly caused by DENV1 (genotype I and genotype V) and DENV2 (Asia I, Asia II, and Cosmopolitan). Furthermore, correlation analysis indicated a significant positive correlation between the number of imported and local cases (correlation coefficient = 0.936). Multiple sequence alignment and phylogenetic divergence analysis revealed that the local isolates are closely related to the isolates from Myanmar and Laos. Interestingly, recombination analysis found that the DENV1 and DENV2 isolates in this study had widespread intra-serotype recombination. Taken together, the results of the epidemiological investigation imply that the dengue outbreak in Yunnan was primarily due to imported cases. This study provides a new reference for further investigations on the prevalence and molecular epidemiology of DENV in Yunnan, China.

Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.

Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor’s gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

RNA interference in cytochrome P450 monooxygenase (CYP) gene results in reduced insecticide resistance in Megalurothrips usitatus Bagnall.

Genes of the cytochrome P450 (CYP450) superfamily are known to be involved in the evolution of insecticide resistance. In this study, the transcriptomes of two Megalurothrips usitatus Bagnall (Thysanoptera: Thripidae) strains (resistant and susceptible) were screened for detoxification genes. MusiDN2722 encodes a protein composed of 504 amino acid residues with a relative molecular mass of 57.3kDa. Multiple sequence alignment and phylogenetic analysis showed that MusiDN2722 is a member of the CYP450 family and has characteristics of the conserved CYP6 domain shared by typical CYP450 family members. RT-qPCR (real-time quantitative polymerase chain reaction) analysis showed that MusiDN2722 was upregulated in the acetamiprid-resistant strain compared with the susceptible strain (p < 0.05), and the relative expression level was significantly higher at 48h after exposure than at 24h after exposure. The interference efficiency of the injection method was higher than that of the membrane-feeding method. Silencing of MusiDN2722 through RNA interference significantly increased the sensitivity of M. usitatus to acetamiprid. Overall, this study revealed that MusiDN2722 plays a crucial role in the resistance of M. usitatus to acetamiprid. The findings will not only advance our understanding of the role of P450s in insecticide resistance but also provide a potential target for the sustainable control of destructive pests such as thrips.

Identification and functional characterization of a new terpene synthase gene from Chrysanthemum indicum var. aromaticum

Terpene synthase (TPS) is a key enzyme in the synthesis of terpenoids in plants and plays an important role in the regulation of the synthesis of aromatic substances in plants. In this study, we treated Chrysanthemum indicum var. aromaticum as the experimental material to clone the CiTPS gene. The open reading frame of CiTPS is 1 818 bp, encoding a putative protein of 605 amino acids. There is a monoterpene synthase conserved domain and a highly conserved sequence, DDXXD, (N,D)D(L,I,V)X(S,T)XXXE and RRX8W, respectively, which belong to the monoterpene synthase family. The multiple sequence alignment and phylogenetic analysis showed that the CiTPS gene belongs to the TPS-b subfamily. The pBI121-TPS-GFP recombinant plasmid and the pBI121-GFP empty vector plasmid were introduced into Agrobacterium by electroporation and transferred into wild-type tobacco by the Agrobacterium-mediated method. Three transgenic CiTPS lines were screened, and the secretions of wild-type tobacco and transgenic tobacco T1 leaves in the vigorous growth period were analysed. The volatiles of the transgenic lines were similar to those of wild-type tobacco, but their monoterpenes increased significantly, and the sesquiterpenoids and diterpenoids decreased or remained unchanged, indicating that the CiTPS gene is related to the synthesis of the monoterpenes.

Comprehensive analysis of the WRKY gene family in Cucumis metuliferus and their expression profile in response to an early stage of root knot nematode infection.

Root-knot nematode (RKN) is a major factor that limits the growth and productivity of important Cucumis crops, such as cucumber and melon, which lack RKN-resistance genes in their genome. Cucumis metuliferus is a wild Cucumis species that displays a high degree of RKN-resistance. WRKY transcription factors were involved in plant response to biotic stresses. However, little is known on the function of WRKY genes in response to RKN infection in Cucumis crops. In this study, Cucumis metuliferus 60 WRKY genes (CmWRKY) were identified in the C. metuliferus genome, and their conserved domains were classified into three main groups based on multiple sequence alignment and phylogenetic analysis. Synteny analysis indicated that the WRKY genes were highly conserved in Cucumis crops. Transcriptome data from of C. metuliferus roots inoculated with RKN revealed that 16 CmWRKY genes showed differential expression, of which 13 genes were upregulated and three genes were downregulated, indicating that these CmWRKY genes are important to C. metuliferus response to RKN infection. Two differentially expression CmWRKY genes (CmWRKY10 and CmWRKY28) were selected for further functional analysis. Both CmWRKY genes were localized in nucleus, indicating they may play roles in transcriptional regulation. This study provides a foundation for further research on the function of CmWRKY genes in RKN stress resistance and elucidation of the regulatory mechanism.

Emergence of allostery through reorganization of protein residue network architecture.

Despite more than a century of study, consensus on the molecular basis of allostery remains elusive. A comparison of allosteric and non-allosteric members of a protein family can shed light on this important regulatory mechanism, and the bacterial biotin protein ligases, which catalyze post-translational biotin addition, provide an ideal system for such comparison. While the Class I bacterial ligases only function as enzymes, the bifunctional Class II ligases use the same structural architecture for an additional transcription repression function. This additional function depends on allosterically activated homodimerization followed by DNA binding. In this work, we used experimental, computational network, and bioinformatics analyses to uncover distinguishing features that enable allostery in the Class II biotin protein ligases. Experimental studies of the Class II Escherichia coli protein indicate that catalytic site residues are critical for both catalysis and allostery. However, allostery also depends on amino acids that are more broadly distributed throughout the protein structure. Energy-based community network analysis of representative Class I and Class II proteins reveals distinct residue community architectures, interactions among the communities, and responses of the network to allosteric effector binding. Bioinformatics mutual information analyses of multiple sequence alignments indicate distinct networks of coevolving residues in the two protein families. The results support the role of divergent local residue community network structures both inside and outside of the conserved enzyme active site combined with distinct inter-community interactions as keys to the emergence of allostery in the Class II biotin protein ligases.